PROJECT SUMMARY/ABSTRACT Age is the biggest risk factor for neurodegenerative diseases such as Parkinson?s disease (PD) and Alzheimer?s disease. Homeostatic decline that occurs as part of aging might promote the accumulation of protein aggregates observed in these diseases, which can affect function and cause cell death. In a subset of diseases called synucleinopathies, the presynaptic protein alpha-synuclein (aSyn) oligomerizes into insoluble amyloid fibrils in neurons and can lead to distinct clinical phenotypes depending on the population affected. Accumulation in the substantia nigra pars compacta (SNc) occurs in PD, resulting in the death of midbrain dopamine neurons and deterioration of motor skills; however, motor dysfunction may actually represent a late stage of the disease. Clinicians observed a prodromal period of idiopathic PD marked by non-motor symptoms. Commonly, patients reported a loss of smell, and assessment of the nasal cavity found aSyn aggregates. This led to the hypothesis that aSyn pathology originates in the periphery before propagating to the brain. However, the nose to brain progression of aSyn pathology has not been directly demonstrated. Additionally, whether age-related factors increase susceptibility to aSyn fibril seeding and spread has not been determined. The goal of this proposal is to establish a training plan to learn and utilize techniques to interrogate neural pathways connecting the periphery to the brain. I will test the hypothesis that age-relevant factors underlie the susceptibility to peripheral synucleinopathy. I will also determine how aSyn pathology affects the olfactory system and progresses from the nose to the brain. Under the guidance of Dr. Viviana Gradinaru and Dr. David Chan, I will advance my technical and professional training, which will prepare me for independent research. In the mentored phase (K99), I will perform a focused analysis of PD-relevant proteins in olfactory pathway structures to see how age and seeding of pathologic aSyn in the nasal cavity affects expression. I will also perform in vivo imaging of mitochondrial dynamics in olfactory regions to visualize the impact of aging and aSyn pathology on cellular physiology. The techniques and results acquired in the mentored phase of the award will facilitate the transition to independent research (R00). In this phase, I will use tissue clearing methods we developed (Bone CLARITY, PACT) to visualize aSyn pathology and olfactory pathways in transparent mouse skulls. Using novel viral capsids that we engineered, I will manipulate aSyn homeostasis-relevant protein expression in the periphery to determine if they contribute to peripheral synucleinopathy pathogenesis. I will also image genetically encoded calcium and dopamine indicators in vivo to understand how peripherally seeded aSyn fibrils progress through the neurocircuitry responsible for olfaction and affect olfactory function. Completing this proposal will contribute to our understanding of synucleinopathy etiology in peripheral systems and its progression to the brain. The findings will be key in developing novel diagnostic and therapeutic strategies for synucleinopathies.